This proposal has three goals. First is to examine Ca++ mobilization responses to nutrients of two existing enteroendocrine cells, STC-1 and GluTag cells. Second is to derive novel reversibly transformed enteroendocrine cell lines and characterize their Ca++ responses to nutrients. Third is to examine in situ Ca++ responses to nutrients by enteroendocrine cells in a transgenic mouse engineered to express the calcium sensor GFP-aequorin. The nutrients to be tested include glucose, fats, peptones, amino acids and some bitter compounds. [unreadable] [unreadable] To accomplish the first goal we will introduce a non-intrusive bioluminescent Ca++ sensor, GFP-aequorin, into STC-1 and GluTag cells. We will then work out the technical details of using this Ca++ sensor to monitor enteroendocrine cell responses to nutrients. For the purposes of the second and third goals we will generate a transgenic mouse line expressing both GFP-aequorin and temperature sensitive SV40 T antigen (tsTag) from the proglucagon promoter that is active in L type enteroendocrine cells that produce glucagon like peptide-1 (GLP-1). Novel transformed L type enteroendocrine cell lines will be isolated from the small and large intestines of these transgenic mice. Growth at the restrictive temperature will inactivate tsTag and cause these cell lines to revert to a non-transformed phenotype more akin to that of native enteroendocrine cells. Ca++ responses to nutrients will be examined in vitro in these "native" L cells. For the final goal we will monitor responses of enteroendocrine L cells in situ in the small and large intestines of these transgenic animals. [unreadable] [unreadable] It is anticipated that results from this work will validate the technical approach of using GFP-aequorin to monitor enteroendocrine cell responses in vitro and in situ. This approach should provide important new information and preliminary data on enteroendocrine cell responses to nutrients. Future R01 proposals based on the present work and the reagents developed here will examine in greater detail and at mechanistic levels the responses of L type enteroendocrine cells in vitro and in situ. [unreadable] [unreadable] This proposal has relevance to GI metabolism, appetite and satiety. The knowledge gained from this proposal may lead to effective interventions for disease states including obesity, anorexia, cachexia and diabetes. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]